ABSTRACT In the US there are ~750,000 heart attacks (acute myocardial infarctions) a year, and ~300,000 patients undergo scheduled cardiac ischemia during cardiac surgery. Beyond reperfusion itself, there are no FDA- approved interventions to limit myocardial injury due to ischemia and reperfusion (IR). This renewal proposal is part of an ongoing program to elucidate mitochondrial & metabolic events in IR and exploit this knowledge to develop small molecule cardioprotective therapies. Our focus is the interplay between acid pH and metabolism in the ischemic heart, based on the following discoveries: (i) SIRT1 is required for cardioprotection and protective metabolic remodeling. (ii) Among the metabolites regulated by SIRT1 is 2- hydroxyglutarate (2-HG), a hypoxic signaling molecule. (iii) We have found a novel mechanism by which SIRT1 can affect metabolism - impacting cardiomyocyte pH via signaling to NHE1. (iv) Acidosis in ischemia is cardioprotective, but the mechanisms are poorly defined. We have discovered key metabolic events in ischemia are triggered by direct effects of acid on metabolic enzymes. (v) We propose 2-HG activates Hypoxia Inducible Factor (HIF) and inhibits the necrosis mediator Alk-B homolog 7 (ALKBH7). (vi) It is thought that reversal of mitochondrial complex II (Cx-II) drives accumulation of succinate, which then drives pathologic ROS generation at reperfusion. However, new data suggest poor consensus on the mechanism of succinate accumulation, its possible roles in ischemia, and its regulation by pH. Overall, we hypothesize that SIRT1 enhances ischemic acidosis, triggering cardioprotective metabolic events including 2-HG and succinate accumulation. This hypothesis will be tested through pursuit of the following specific aims? Aim 1 will investigate the mechanism by which SIRT1 enhances ischemic acidosis. Aim 2 will investigate mechanisms by which acid and 2-HG signal cardioprotection. Aim 3 will investigate the mechanism(s) of ischemic succinate accumulation and the timing of Cx-II inhibition for therapeutic benefit. These studies will use adult cardiomyocytes, perfused hearts, the in-vivo LAD occlusion model of IR injury, and engineered mice including Alkbh7-/- and cardiac specific Sirt1-/-. We will also employ novel pharmacologic agents (Cx-II inhibitors and cell-permeable 2-HG analogs), fluorescent pH imaging, LC-MS/MS based metabolomics, and 13C dynamic labeling metabolomics. This work will advance fundamental knowledge on ischemic cardiac metabolism, will develop small molecule therapies, and will offer mechanistic insight applicable to multiple tissues and pathologies.